In recent years, the number of product recalls and contamination incidents involving pathogenic bacteria has significantly increased, and the ensuing infections continue to be an ongoing problem for public health and agriculture. Due to the widespread impact of these pathogens, there is a critical need for rapid, on-site assays that can provide rapid results. In this work, we demonstrate the development of a rapid and simple test based on the combination of reverse transcription with recombinase polymerase amplification followed by lateral flow strip detection of viable Escherichia coli O157:H7 cells by detecting the RNA of the pathogen. The optimized method can be performed for approximately 2 h with a detection limit of 10 CFU/mL of E. coli O157:H7 in buffer, spinach, and ground beef samples. Our assay is sensitive, detecting only E. coli O157:H7 and not nonpathogenic E. coli or other similar pathogens. This strategy was able to distinguish viable from nonviable bacteria and more significantly was able to detect viable but nonculturable bacteria, which is a major issue when using culture-based methods for monitoring pathogenic bacteria. An important advantage of this test is that it can provide timely identification and removal of contaminated consumables prior to distribution without an extensive sample preparation.
Enteric fever continues to be a major cause of mortality and morbidity globally, particularly in poor resource settings. Lack of rapid diagnostic assays is a major driving factor for the empirical treatment of enteric fever. In this work, a rapid and sensitive method ‘Miod’ ‘has been developed. Miod includes a magnetic nanoparticle-based enrichment of target bacterial cells, followed by cell lysis and loop-mediated isothermal amplification (LAMP) of nucleic acids for signal augmentation along with concurrent measurement of signal via an in–situ optical detection system. To identify positive/negative enteric fever infections in clinical blood samples, the samples were processed using Miod at time = 0 hours and time = 4 hours post-incubation in blood culture media. Primers specific for the STY2879 gene were used to amplify the nucleic acids isolated from S. typhi cells. A limit of detection of 5 CFU/mL was achieved. No cross-reactivity of the primers were observed against 106 CFU/mL of common pathogenic bacterial species found in blood such as E. coli, P. aeruginosa, S. aureus, A. baumanni, E. faecalis, S. Paratyphi A and K. pneumonia. Miod was tested on 28 human clinical blood samples. The detection of both pre-and post-four-hours incubation confirmed the presence of viable S. typhi cells and allowed clinical correlation of infection. The positive and negative samples were successfully detected in less than 6 hours with 100% sensitivity and specificity.
BackgroundThe temporary anchorage devices (TADs) which include miniscrew implants (MSIs) have evolved as useful armamentarium in the management of severe malocclusions and assist in complex tooth movements. Although a multitude of factors is responsible for the primary and secondary stability of miniscrew implants, contemporary research highlights the importance of biological interface of MSI with bone and soft tissue in augmenting the success of implants.The inflammation and remodeling associated with MSI insertion or loading are reflected through biomarkers in peri-miniscrew implant crevicular fluid (PMICF) which is analogous to the gingival crevicular fluid. Analysis of biomarkers in PMICF provides indicators of inflammation at the implant site, osteoclast differentiation and activation, bone resorption activity and bone turnover. The PMICF for assessment of these biomarkers can be collected non-invasively via paper strips, periopaper or micro capillary pipettes and analysed by enzyme-linked immunosorbent assay (ELISA) or immunoassays. The markers and mediators of inflammation have been previously studied in relation to orthodontic tooth movement include interleukins (IL-1β, IL-2, IL-6 and IL-8), growth factors and other proteins like tumour necrosis factor (TNF-α), receptor activator of nuclear factor kappa-B ligand (RANKL), chondroitin sulphate (CS) and osteoprotegerin (OPG).Studies have indicated that successful and failed MSIs have different concentrations of biomarkers in PMICF. However, there is a lack of comprehensive information on this aspect of MSIs. Therefore, a detailed review was conducted on the subject.ResultsA literature search revealed six relevant studies: two on IL-1β; one on IL-2, IL-6 and IL-8; one on TNF-α; one on CS; and one on RANKL/OPG ratio. One study showed an increase in IL-1β levels upon MSI loading, peak in 24 hours (h), followed by a decrease in 21 days to reach baseline in 300 days. A 6.87% decrease in IL-2 levels was seen before loading and a 5.97% increase post-loading. IL-8 showed a 6.31% increase after loading and IL-6 increased by 3.08% before MSI loading and 15.06% after loading. RANKL/OPG ratio increased in loaded compared to unloaded MSIs.ConclusionsCytokines (mainly ILs and TNF-α) and RANKL/OPG ratio showed alteration in PMICF levels upon loading of MSIs as direct or indirect anchorage.
Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardio-degenerative disorder caused by decreased expression of frataxin, a protein that localizes to mitochondria and is critical for iron-sulfur-cluster (ISC) assembly. There are no proven effective treatments for FRDA. We previously screened a random shRNA library and identified a synthetic shRNA (gFA11) that reverses the growth defect of FRDA cells in culture. We now report that gFA11 decreases cytokine secretion in primary FRDA fibroblasts and reverts other changes associated with cell senescence. The gene-expression profile induced by gFA11 is remarkably similar to the gene-expression profile induced by the p38 MAPK inhibitor SB203580. We found that p38 phosphorylation, indicating activation of the p38 pathway, is higher in FRDA cells than in normal control cells, and that siRNA knockdown of frataxin in normal fibroblasts also increases p38 phosphorylation. Treatment of FRDA cells with p38 inhibitors recapitulates the reversal of the slow-growth phenotype induced by clone gFA11. These data highlight the involvement of the p38 MAPK pathway in the pathogenesis of FRDA and the potential use of p38 inhibitors as a treatment for FRDA.
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